Antibiotic complex taimycine and method for the preparation thereof

ABSTRACT

A MICROBIOLOGICAL PROCESS FOR THE PREPARATION OF THE NEW ANTIBIOTIC SUBSTANCE TAIMYCINE AND ITS SALTS CONSISTING OF TAIMYCINE A, TAMIYCINE B, TAMIYCINE C. THE PROCESS IS CHARACTERIZED IN THAT THE NEW MICROORGANISM STREPTOMYCES MICHIGANENSIS VAR. AMYLOLYTICUS IS CULTIVATED IN AEROBIC CONDITIONS IN A LIQUID CULTURAL MEDIUM CONTAINING A CARBON SOURCE, NITROGEN SOURCE AND MINERAL SALTS AT A TEMPERATURE OF FROM 24* TO 37*C. OVER A PERIOD OF TIME OF FROM 72 TO 160 HOURS AT A PH OF FROM 6 TO 8 AND THAT THE ANTIBIOTIC COMPLEX THUS OBTAINED IS ISOLATED FROM THE FERMENTATION BROTH BY EXTRACTON WITH SUITABLE SOLVENTS AND SUCCESSIVE PRECIPITATION AND THE ANTIBIOTIC COMPLEX MAY BE SEPARATED INTO THE THREE COMPONENTS TAIMYCINE A, TAMIYCINE B AND TAIMYCINE C. A NEW ANTIBOTIC SUBSTANCE CALLED TAIMYCINE AND ITS SALTS HAVING ANTIHELMINTIC AND ANTIPROTOZOAL ACTIVITY AND ITS THREE ACTIVE ANTIBIOTIC COMPONENTS CALLED TAIMYCINE A, TAIMYCINE B AND TAIMYCINE C ARE ALSO DESCRIBED. THE MICROORGANISM HAS BEEN DEPSITED AMONG OTHERS AT THE INSTITUTE OF MICROBIOLOGY OF RUTGERS UNIVERSITY RECEIVING THE INDEX NUMBER I.M.R.U. 3932 AND AT THE INSTITUTE OF PLANT PATHOLOGY AT THE UNIVERSITY OF MILAN (ITALY), RECEIVING THE INDEX NUMBER I.P.V. 1953.

PERCENT TRANSMITTANCE Feb. 22, 1972 Filed July 21', 1970 F I G I TAIMYCINE 200 24'0 2550 200 :00 300 350 al'so 300 my ULTRAVIOLET ABSORPTION SPECTRA OF TA MYCIN ES IN METH ANOL 1FIG.2

WAVE LENGTH I MICRON) 2.5 a 4 s s 7 e 9 1o 11 12 13 14 15 100 I I I I- I I I I I I T T I I 'I I I I I I I I I I I I I I I I I I I I o L 4000 3500 3000 2500 2000 1900 100% [.R SPECTRA OF ANTIBIOTIC 2 I800 I500 1400 1300 1200 I100 1000 900 800 700 NUMBER OF WAV 6I-AB I IN KBr I TAIMYCINE A PERCENT TRANSMITTANCE PERCENT TRANSMITTANCE 1972 G. CASSINELLI ET AL 3,644,619

ANI'IUIOTIC COMPLEX TAIMYCINE AND METHOD FOR THE PREPARATION THEREOF Filed July 21, 197C 2 Sheets-Sheet 2 WAVE LENGTH (MICRON) I l I I I I I I I I I I I l I 1I I I l I 1 I I 0 l l l I l l l l I l l NUMBER OF WAVES (CM-I IR. SPECTRA OF ANTIBIOTIC 2461-52 (IN KBrI=TA|MYClNE B WAVE LENGTH (MICRON) I l I II I I I I l I I II I l l I I l l l 0 1 I l l I l l l 1' I l I I I l I 1 4000 3500 3000 2500 2000 1900 1800 1700 1500 1500 1400 1300 1200 1100 1000 900 800 700 NUMBER OF WAVES (CM-0 I.R. SPECTRA-OF ANTIBIOTIC 2461 C (IN KBr) TAIMYCINEf-C o 3,644,619 [Ce Patented Feb. 22, 1972 I.M.I. 131,670, and at the Institute of Microbiology of 3,644,619 Rutgers University (U.S.A.) receiving the index number ANTIBIOTIC COMPLEX TAIMYCINE AND METH' I.M.R.U. 3932, and at the Institute of Plant Pathology FOR PREPARATION THEREOF at the University of Milan (Italy) receiving the index Gmseppe Cassmelh.Rlvanazzanopawaand l 5 number I.P.V. 1953. At the last named institute, the wg ggg gg gg gg gg ggzzfig h g microorganism is freely available. The microorganism Mimi], Italy Streptomyces michiganensis var. arnylolyticus has been Continuation-impart of application Ser. No. 734,875, lsolated from a soil sample taken in Trang (Thailand).

June 6, 1968. This application July 21, 1970, Ser. It shows the following morphological, cultural and bio- No. 56,773 chemical properties, Claims priority, application Italy, June 6, 1967,

1 923 7 Morphologlcal properties CL A611 21/00 On the usual culture media, the vegetative mycelium 424-421 11 Cla'ms consists of hyphae, of 0.5-0.9/L thickness, long and 15 abundantly branched, which form thicker hyphae having a thickness of 1.1-1.4 collected in bunches sym- ABSTRACT OF THE DISCLOSURE podially branched. The latter are rather long, straight A microbiological Process for the Preparation of the or flexuous and form conidia which are initially disposed new antibiotic substance Taimycine and its salts consistin hai and then free. The onidia are oval, have a,

ing of Taimycine A, Taimycine B, Taimycine C. The smooth surface and a size of from 1.1 to 1.4 x 1.2-1.9/L- process is characterized in that the new microorganism Streptomyces michiganensis var. amylolyticus is culti- Cultural and biochemical properties vated in aerobic conditions in a liquid cultural medium Table 1 lists the cultural properties obtained on difcontaining a carbon source, nitrogen source and mineral ferent media, culturing the microorganism at 28 C. and salts at a temperature of from 24 to 37 C. over a 25 carrying out the observations on the 3rd, 8th, 15th and period of time of from 72 to 160 hours at a pH of from 20th day after inoculation. The microorganism shows a 6 to 8 and that the antibiotic complex thus obtained quick and abundant development with the formation of is isolated from the fermentation broth by extraction with a compact vegetative mycelium, rather solid with a suitable solvents and successive precipitation and the smooth patina on synthetic media, a slightly folded patina antibiotic complex may be separated into the three comon organic media. On synthetic media, the color of the ponents Taimycine A, Taimycine B and Taimycine C. vegetative mycelium and of its back-side is from straw- A new antibotic substance called Taimycine and its salts yellow to lemon-yellow while on organic media it is ochre having antihelmintic and antiprotozoal activity and its and light chestnut-brown. On organic media, there is also three active antibiotic components called Taimycine A, produced a light tea-colored soluble pigment which Taimycine B and Taimycine C are also described. The changes to a light chestnut-brown tonality on aging. The microorganism has been deposited among others at the aerial mycelium is abundant on all cultural media, having Institute of Microbiology of Rutgers University receiving a rather pulverous aspect. It is ivory-white colored with the index number I.M.R.U. 3932 and at the Institute of sulfur-yellow tonalities on synthetic media, while on Plant Pathology at the University of Milan (Italy), reorganic media this color changes from chalk-white to ceiving the index number I.P.V. 1953. ivory-white With tonalities sometimes light beige, sometimes attenuated sulphur-yellow. The biochemical properties of the microorganism under examination are:

This is a continuation-in-part application of Ser. No. 734,875, filed June 6, 1968, now abandoned.

Our invention relates to an antibiotic complex Taimycine which includes the substances Taimycine A, Taimycine B, Taimycine C and salts thereof, which have proved useful in therapy as antihelmintic and antiprotozoal agents. The invention includes a process for the preparation of this antibiotic complex by cultivation of a Taimygelatinzrapid total hydrolysis nitrateszare reduced to nitrites starchzdiscrete hydrolysis milkzcoagulation and peptonization melanine light production tyrosine:rapidly decomposed production of hydrogen sulphide:modest cine-producing strain of the hitherto unknown micro- The microorganism utilizes: l-arabinose, glucose, dorganism Streptomyces michiganensis var. amylolyticus xylose, d-fructose, maltose, d-mannitol and mesoinositol. which we also call Streptomyces F1. 2461. Streptomyces It does not utilize: saccharose, ramnose and rafiinose. The michiganensis var. amylolyticus has been deposited at microorganism does not grow at 50 C. and does not the Commonwealth Mycological Institute, Ferry Lane, produce sclerotia. In submerged stirred liquid culture, the Kew, Surrey (Great Britain) receiving the index number strain produces the antibiotic complex Taimycine.

TABLE 1.-CULTURAL PROPERTIES Soluble Medium Growth Aerlal mycellum Vegetative mycelium pigments Bennets agar Abundant; in smooth patina Abundant, from ivory cream to From straw-yellow to lemon-yellow Light tea color.

light belge with yellow-sulphur to light chestnut-brown with idenflat tonalities. tica-l back-side. Czapecks agar Abundant Abundant, from White to ivory; flat. Scanty, colorless Asparagine glucose Abundant; in smooth patina Abundant, fiat; from ivory-white to Also the backside from straw-yellow agan pale yellow-sulphur. to lemon-yellow. Glycerine glycine do Abundant; flat ivory color with From straw-yellow to lemon-yellow agarJ beige tonalities, sometimes pale with sometimes brown tonalities;

pink-vinous. identical back-side. Emerson's agar Abundant; in patina more or less Abundant; flat, from chalk-white to Chestnut-brown colored; identical- Light chestnutrelieved. beige-white. back-side. brown color. Starch agar and Abundant; in smooth patina Abundant; fiat; yellow-sulphur From lemon-yellow to light ochresalts. yellow; identical back-side. Potato agar 3 do l Abundant; flat, ivory-whitc with From straw-yellow to ochre-yellow Tea color.

beige tonalities. to light chestnut-brown; identical back-side.

TABLE 1.Continued Soluble Medium Growth Aerial mycelium Vegetative mycelium pigments Oats agar 4 do From ivory-white to sulphur-yellow From straw-yellow to lemon-yellow abundant, flat. to light-brown: identical backside. Glycerinedo From ivory-white to clear snlphur- From straw-yellow to lemon-yellow;

asparagine agar 1 yellow; abundant, flat. identical backside. Yeast-extract Abundant; in patina more or less From chalk-white to light-biege More or less dark chastnut-brown; Chestnut-brown glucose agar. relieved. with sulphur-yellow tonalities; identical back-side. color.

abundant, flat. Peptone-starch Abundant; in smooth patina From chalk-white to beige, to Light chestnut-brown with identical Do.

agar. sulphur-yellow; abundant, flat. back-side. Peptone agar po- .do Abundant; flat, chalk-white color--- From straw-yellow to light chestnut- Tea color.

tassium nitrate. brown with identical backside.

1 Waksman, S. A; The Actinornycetes, vol. II, The William and Wirlkins Co. 1961, p. 328434.

2 Pridham, T. G., Anderson, P., Foley, 0., Lindenielser, L. A., Hesseltine, M., and Benedict, R. B.: Antibiotics Annual, 1956-1957, p. 947-953.

3 To 200 g. of potatoes (boiled and filtered through gauze) 20 g. of glucose and 20 g. of agar are added. Then the mixture is taken up to a. volume of 1 liter and sterilized at 120 C. for 20 minutes.

4 Baldacci, E., Giolitti, G., Kiistcr, E., and Scotti, T.: Journal of Microbiology 1961, 9, p. 39.

Identification of e Strain TABLE 3.BIOCHEMICAL PROPERTIES The properties of the microorganism described above 20 relate it to the genus Streptomyces Waksman and Henrici F I. 14970 (Bergeys Manual of Determinati've Bacteriology, 7th ed., 1957, p. 744-745). The strain belongs to the section Utilization p. 191). A comparison between the properties of the Hydrolysis of starch Production of hydrogen sulphide. microorganism under examination and those of the species Reduction min-ates belonging to the cited systematic groups (Taxa) has and Peptomzamo Oimilk shown a close resemblance between the microorganism 3r under examination and Streptomyces mzchzganenszs, Cor- AS a result of the above comparison of the biochemical baz et (Arch' Mikrobiol" 1957 205) Tables properties of Streptomyces RI. 2461 and Streptomyces Rectus flexibilis series olive-buff of Pridham et a1. 1.31 330%" i i (appl. Microbiol. .1958, 6, p. 52), to the series virgatus 25 i i of Baldacci (Giornale d1 Microbiologia, 1918, 6, p. 10), l/i i l to n to the series Erythrochromogenes of Waksman (The ,51 ,3 1; i 1' Actinomycetes, vol. II, 11961, p. 117 and p. 149), and to gg z I the group of streptomycetes having griseus aerial my- Hydrolysis of g elatin cclium of Hiitter (Giornale di Microbiologia, 1963, 11, 30 figgffiggggfifi a E 1,:

2 and 3 g comparative data of the microorganism michiganensis strain 14970 ATCC, we conclude that the under examination and the last-mentioned species. The microorganism streptomyces F 2461 belongs to h differences between the two cultures are in variations of 40 pecies Streptomyces i hi i The difi ren es tonality in the vegetative mycelium color, in the aerial pointed out make it possible to consider the strain F.I. mycelium color, and starch utilization. 2461 a variety of this species for which we propose the TABLE 2.CULTURAL PROPE RTIES Streptomyces F.I. 2461 Strcptomyces michiganensz's strain 14970 ATCC Medium Vegetative niycelium Aerial myceliinn Vegetative mycelium Aerial mycelium Bennets agar From straw-to lemon-yellow until Light-beige with sulphur-yellow From straw-to lemon-yellow until Beige with sulphurlight chestnut-brown; tcatonalities; abundant. light chestnut-brown; teayellow tonalities; colored soluble pigment. colored soluble pigment. abundant.

Czapeck s agar Colorless Fair white Colorless Fair white.

Asparagine glucose From straw-to lemon-yellow From ivory-white to clear beige; From straw-to lemon-yellow Ivory-white;

agar. abundant. abundant.

.Asparag ne Lemon-yellow Beige; abundant Lemon-yellow Beige; abundant.

glycerinc agar.

Emerson's agar. Chestnut-bi'own, soluble pigment Beige; abundant Chestnut-brown; chestnut brown Beige with sul hurchestnut-brown. soluble pigment. yellow ton a ties;

abundant.

Starch agar and From straw-to lemon-yellow Froin beige to pale sulphur- From lemon-yellow to orange- From beige to clear sal yellow; abundant. yellow; soluble pigment lemonsulphur-yellow;

yellow colored. abundant.

Potato agar From straw-yellow to ochre to Ivory-white with beige tonalities; From straw-yellow to ochre to Ivory-white with light chestnut-brown; tea abundant. light chestnut-brown; tea beige tonalities; colored soluble pigment. colored soluble pigment. abundant.

o agar Straw-yellow to lemon-yellow. Ivory-white with beige tonalities From lemon-yellow to orange- From beige-to sulfur- V sometimes sulphur-yellow; yellow; lemon colored soluble yellow; abundant.

abundant. pigment.

g i parado Ivory-white to sulphur-yellow; Straw-yellow to lemon-yellow-.." Ivory-white to sulgine agar. I abundant yellow; abundant.

Yeast-glucose ex- Light chestnut-brown; chcstnut- From chalk-white to light beige; Light chestnut-brown; chestnut- Beige with clear t t agar. brown soluble pigment. abundant. brown soluble pigment. sulphur-yellow tlonazlities; abunan Potassium nitrate- From straw-yellow to light Chalk-white, abundant From straw-yellow to light Absent.

peptone agar. chestnut-brown. chest nut-brown.

Tyrosine agar (Gov Production of chestnut soluble pigment. Quick decomposition of Production of a black soluble pigment. Slow decomposidon et al., 1955) l tyrosine. tion of'tyrosine Tyrosine agar Identical behavior of both the strains with slight production of a melanoide pigment.

(Waksman, 1061).

1 Gordon, RE. and Smith M.M..T. Bact. 1955 69, 147.

denomination Streptomyces michiganensis var. amylolyticus. Hiitter (1963) has shown that strains belonging to the species Streptomyces michz'ganansis may produce various antibiotics having antibacterial and antifungal activity; however, the production of substances having antihelmintic activity is not known. Examination carried out by us has confirmed that the mycelium extract of the strain 14970 ATCC of Streptomyces michiganensis does not contain substances having antihelmintic activity.

The microorganism Streptomyces michiganensis var. amylolyticus or F1. 2461 may be stored by sequential transfers on solid media or by lyophylization of a suspension of spores in milk.

The invention provides a microbiological process for the preparation of the antibiotic complex Taimycine which comprises fermentation of a Taimycine-producing strain of the microorganism Streptomyces michigagnensis var. amylolyticus in a liquid culture medium containing an assimilable source of carbon, an assimilable source of nitrogen and mineral salts. The microorganism is preferably grown in a liquid cultural meidum under aerobic conditions at from 24 C. to 37 C., preferably 28 C., for from 72 to 160 hours. The pH may vary from 6 to 8 according to the fermentation medium used.

As a carbon source glucose, dextrin, starch, various meals (of Indian maize, soya or wheat, for example), corn steep and other substances may be used. The nitrogen source, besides the above-mentioned complex substances containing nitrogen, may for example be casein, a casein hydrolyzate, cotton-seed meal and ammonium salts such as a sulphate, phosphate, chloride or other substance. The mineral salts vary according to the medium employed. Calcium carbonate is nearly always present and the chlorides, sulphates, phosphates, for example, of sodium, potassium, magnesium, manganese, iron, copper, zinc and cobalt may be added. The fermentation may be carried out in Erlenmeyer fiasks or in laboratory or industrial fermenters of various capacity.

The antibiotic complex Taimycine comprises three substances called Taimycine A, Taimycine B and Taimycine C, which show similar physico-chemical properties but different biological activity. The determination of the concentration of the antibiotic complex in the culture medium can be carried out during the growth of the microorganism by testing samples obtained by extracting the moist mycelium with methyl alcohol. After evaporation of the solvent in vacuo, the residue is taken up with the smallest possible volume of dimethylformamide and then diluted with water. Such samples are tested on microorganisms sensitive to the antibiotic, for example Rhabditis macrocerca, by preparing a series of dilutions of the sample under examination and comparing them with solutions of the antibiotic complex of known titer. Samples obtained from subsequent purifications may be titrated analogously. Alcoholic solutions of samples partially purified may be spectrophotometrically titrated at 290 and 320 me in comparison to samples of the components of the complex of known titer.

When the fermentation is over, the antibiotic complex mostly contained in the mycelium can be extracted with an organic solvent such as a lower alcohol, acetone, chloroform, methylene dichloride or methylisobutylketone. Methyl alcohol is employed in practice. After three extractions carried out each with a volume which is /3 of that of the culture, the extracts are collected and concentrated under reduced pressure to from /6 to /s of the original volume. The resulting precipitate which contains 10% of the antibiotic is filtered and centrifuged. The supernatant, adjusted to pH 5.8-6.2, is extracted with petroleum ether to remove impurities. The lower layer is extracted with chloroform, the extract washed with water is dried over anhydrous sodium sulphate, concentrated under reduced pressure and precipitated with petroleum ether or with hexane. The crude precipitate contains 10-15% of antibiotic. These crude products are dissolved in chloroform or in a mixture of chloroformzcarbon tetrachloride:methyl alcoholzwater. The solution is adsorbed over a chromatographic column of silicious acid suspended in chloroform. The column is washed with chloroform to remove impurities and then eluted with chloroform containing increasing percentages of ethyl alcohol. The elution is carried out with analysis of the U.V. spectrum of samples of the various fractions and by thin layer chromatography. By eluting with chloroformethanol 3:1) Taimycine C is obtained; by eluting with chloroform-ethanol (2:1) Taimycine B is obtained; by eluting with increased percentages of ethanol Taimycine A containing variable percentages of Taimycine'B is obtained. By diluting the concentrated eluates with water and adjusting the solution to pH 6, partially purified products are recovered. By slow evaporation of the remaining alcohol, the sodium salts from the aqueous concentrates precipitate. Taimycine C may be further purified by crystallization from methanol-water. Taimycine A and B are purified by countercurrent distribution using respectively chloroform-carbon tetrachloride-methyl alcoholwater (3:2:4g1) and (2.5:2.5:4:1) mixtures. They precipitate from. aqueous solutions at pH 6-6.2 as sodium salts. On the average 40 mg. of Taimycine A, 15 mg. of Taimycine B and 5 mg. of Taimycine C per liter of culture broth are obtained.

Physico-chemical properties The substances Taimycine A, B and C have been isolated as sodium salts in the form of white amorphous powders which are stable at neutral or weakly basic pH. They are soluble in dimethylformamide, dimethylsulphoxide, and dioxane, in aqueous alcohols and aqueous acetone; sparingly soluble in lower alcohols, chloroform, acetone, benzol; and practically insoluble in water and petroleum ether. The main physico-chemical properties are given in Table 4.

TABLE 4.PHYSICO-CHEMICAL PROPERTIES Property Taimycine A Taimycine B Taimycine O Melting point 250" C. 260 C. 240" C.

c 0.9 dioxane +7 +415 -12 [alnzaoic 0.77 methanol +24 Percent Carbon 3O 67.26 62 50 Hydrogen. Nitrogen" Sodium U.V. spectrum A CH OH max 240 and 294 my 238 and 321 mp 240 and 290 m 1221. 192. 832. 449 93... 152 and 149). and 255). and 157). AmM.NaOH 0.05 N in CHBOH. 244 and 292 m 235 and 820 m 246 and 287 11111 3 200 502.470 (Erfi'n. 1 4 and and 250). and 156). )\mnx'HC10.05 N in CHQOH 322.5 m 322.5 mp N 0 peaks.

( IZA. 50 1 31. Boo).

With decomposition.

ACHSOH max.

U.V. Spectrum 322 (Elfin, 306) TABLE 5.REACT1ONS Rhabdz'tis macrocera kept in cultures in the laboratory has been cultivated in agar treated with the components of the antibiotic Taimycine in various concentrations.

Syp-hacia ob'velata and Hymenolepis nana, taken from experimentally infected mice, have been immersed in saline solutions buffered at pH 7.5. The components of the antibiotic Taimycine were added at diflerent concentrations. The extent of survival of the two helmintes has been tested at diflerent contact periods. Minimum Im- Taimy- Taimy- Taimy- Reagent OineA cineB Cine mobilizing Dose means the smallest quantity of the sub- Fgmchcmondeu stance capable of immobilizing the movement of the g iteigiiggfimlorideu i I tested organism in 'vitro. In Table 8 are the Minimum mam Inhibiting Dose and the Minimum Immobilizing Dose o e; salt solubleinchlorodetermined after 1 and 4 hours for the separated fractrons of the antibiotic complex Taimycine.

TABLE 8 Taimycine A Taimycine B Taimycine 0 Minimum im- Minimum im- Minimum immobillzmg mobilizing mobilizing dose, 'y/cc. dose, /00. dose, -y/cc.

In 1 In 4 MID, In 1 In 4 MID In 1 In 4 Tested species hour hours 'y/cc. hour hours -y/cc. hour hours Rhabditis macrocerca 5 250 Syphacta obpelata 1,000 1,000 l,000 1,000 1,00 0 l,00 0 Hymenolepis nana 1,000 100 1,000 100 1, 000 1, 000

N Urn-Explanation: +=not determined; +=Not determinable.

Taimycines do not react with the following reagents: ninidrin, nitroprussate, Molish, Fehling, Bial, Anthrone, Orcinol, Ehrlich and Sakaguchi. On the grounds of these physico-chemical properties, Taimycine B especially shows some analogies with the antibiotic Streptolidigine (Waksman and LechevalierThe Actinomycetes 3, 1962, p. 371). It clearly differs from this in biological activity and chromatographic properties which are listed in Table 6.

The antihelmintic activity has been tested in vitro on Albino mice with a body weight of g. and experimentally infected with Hymenolepis nana. The experiments have been carried out on groups of 10 mice each. Results show that 100% of the mice infected with Hymenolepis nana at the 25th day after the infection are cured by administration of different fractions of the antibiotic Taimycine by gastric tubing at doses clearly lower TABLE 6.CHROMATOGRAPHIG PROPERTIES (THIN LAYER) Taimy- Taimy- Taimysodium 3 cine cine cine strepto- Layer Solvent; A B C ligidine MN Kieselgel o 254 o. 40 o. 0. 70 0.85

Kieselgel G Butyl alcohol-acetone-water (4:5:1) 0. 60 0.30 0. 75 0. 80

Kieselgel HF 254366 0. 0. 25 0. 50 0. 85

MN Kieselgel G 254 Ethyzl :gczigatepyridine-isopropyl alcohol-water 0.50 O. 50 0. 80 1. 00

Kieselgel G Propyl alcohol-ethyl acetate-water (8:1:1). 0. 0. 25 0. 0.60

Alumina Butyl aleohol-pyridine-water (6:4:3) 0. 50 0.30 0. 70

Antiprotozoal activity than permitted by the toxicity of the fraction. In Table 9 TABLE 7 Antiprotozoal activity in vitro Tested fraction: MID y/cc. Taimycine A 6.25 Taimycine B 0.78 Taimycine C 25 Antihelmintic activity The antihelmintic activity has been tested in vitro on different helmintes by diiferent techniques:

are the acute toxicity figures (LD for mice.

TABLE 9.ACUTE TOXICITY Administration route Taimycine A Taimycine B Taimycine 0 Oral 1, 000 250 1, 000 Intraperitoneous 25 5 250 Taimy- Taimy- Taimy- Infected bycine A cine B cine C Hymenalepis mma 25 25 50 The invention includes pharmaceutical compositions containing one or more antibiotics according to the invention in admixture with a vehicle, for example a diluent or carrier.

The following examples serve to illustrate the invention without limiting the same:

EXAMPLE 1 Two 300 cc. Erlenmeyer flasks were prepared, each containing 60 cc. of the medium:

Percent Dextrin 3 Casein 0.5 Calcium carbonate 0.4 Ammonium sulphate 0.1

Potassium dihydrogen monophosphate 0.0 Corn steep liquor 0.3 Tap water to 100.

Sterilization was performed by heating at 120 C. for 20 minutes. The pH of the medium after sterilization was from 6.8 to 7. Each flask was inoculated with 0.5 cc. of a spore suspension obtained by washing, with 5 cc. of sterile distilled water, the patina of a day old slant culture of Streptomyces F. I. 2461 developed on glucose potato agar. The flasks were incubated at 28 C. for 24 hours on a rotary shaker with a stroke of 3 cm. at 225 r.p.m. Thereafter 2 cc. of this culture were used each to inoculate a 300 cc. flask containing 60 cc. of the productive medium:

Percent Soluble starch 12 Corn steep liquor 2.5 Calcium carbonate 1 Ammonium sulphate 1 Cotton-seed meal 0.4 Manganous sulphate 0.01 Cobalt chloride 0.0007 Tap water to 100.

The pH was adjusted to 6.2 with 4' N sodium hydroxide solution. Sterilization was carried out by heating at 120 C. for minutes. The culture was incubated at 28 C. as previously described for the vegetative culture. After 120 hours of incubation, the production of 100 g. per cc. of culture broth of the antibiotic complex was obtained.

EXAMPLE 2 The operation was as in Example 1 with the difference that for the productive phase the following medium was employed:

Percent Dextrin 7 Cotton-seed meal 1 Corn steep liquor 1 Ammonium sulphate 0.6 Calcium carbonate 0.5

Potassium dihydrogen monophosphate 0.001 Tap water to 100.

Sterilization was carried out by heating at 120 C. for 20 minutes. The pH after sterilization was from 6.6 to 6.8. The culture was incubated at 28 C. under the conditions of Example 1. After 120 hours of incubation, the production of 100 g. per cc. of culture broth of the antibiotic complex was obtained.

EXAMPIJE 3 Two 300 cc. Erlenmeyer flasks were prepared each containing 60 cc. of the medium:

Percent Dextrin 4 Calcium carbonate 0.5 Ammonium sulphate 0.2 Casein 1 Corn steep liquor 1 Potassium dihydrogen monophosphate 0.001

Tap water to 100.

Sterliziation was carried out by heating at 120 C. for 20 minutes. The pH of the medium after sterilization was from 6.7 to 7. Each flask was inoculated with 0.5 cc. of a spore suspension obtained by washing, with 5 cc. of sterile distilled water, the patina of a 15 day old slant culture of Streptomyces El. 2461 developed on glucose potato agar. The flasks were incubated at 28 C. for 24 hours under the same conditions as in Examples 1 and 2. Thereafter 2 cc. of a culture thus obtained Were used to inoculate 300 cc. flasks containing each a productive medium having the same composition as that employed for the vegetative phase above. The flasks were incubated at 28 C. as described for the vegetative phase. After 120 hours, the concentration of the antibiotic complex per cc. of culture broth was 120 g.

EXAMPLE 4 The operation was as in Example 3 with the difference that for the productive phase the following medium was employed:

Percent Corn steep liquor 1.5 Ammonium sulphate 0.4 Calcium carbonate 0.6 Soluble starch 2 Glucose 0.5 Maize meal 0.1 Soya meal 0.4 Potassium monohydrogen diphosphate 0.0 Bacon-fat oil 0.2.5

Tap water to 100.

The pH was adjusted to 6.7 with 4 N sodium hydroxide solution. The culture was sterilized by heating at 120 C. for 20 minutes. The concentration of the antibiotic complex per cc. of culture broth after 120 hours was 150 g.

EXAMPLE 5 Three 300 cc. flasks each containing 60 cc. of the cultural medium:

Percent Dextrin 4.0 Casein 1.0 Ammonium sulphate 0.1 Corn steep liquor 1.0 Calcium carbonate 0.5

Potassium dihydrogen monophosphate 0.01 Tap water to 100.

were sterilized by heating at 120 C. for 20 minutes and then inoculated with a spore suspension of a culture on glucose potato agar (as in Example 1) of Streptomyces michiganensis var. amylolyticus. The flasks were then incubated for 24 hours at 28 C. on a rotary shaker at 225 rpm. A 10 liter fermenter containing 6 liters of the cultural medium described above was inoculated with the contents of three flasks. This culture was incubated at 27 C. with stirring at 400 g. per minute and introduction of 1 liter sterile air per 1 liter of culture per minute. After 20 hours, 2.5 liters of the culture thus obtained were used to inoculated an liter fermenter containing 50 liters of a medium consisting of:

'Percent Dextrin 6.0 Casein 2.0 Corn steep liquor 1.0 Calcium carbonate 0.5

Potassium mono-phosphate 0.0 Tap water to 100.

Sterilization was carried out by heating at 120 C. for 30 minutes. Fermentation was carried out at 27 C. with a stirring of 250 rpm. and with an air stream of 0.7 liter per liter medium per minute. The fermentation lasted for 110 hours. The maximum concentration was g. of antibiotic complex per cc. of culture broth.

The culture liquid from the fermentation was filtered through a silicious adsorbent. Thus a cake and a filtrate, which was discarded, were obtained. The cake was washed with water, then suspended in methyl alcohol and stirred. The extract was filtered and the extraction was repeated twice. The collected methanol extracts were concentrated under reduced pressure to about liters. The yellow-green precipitate was separated by centrifugation, then washed with water and dried under .vacuum over phosphoric anhydride. 25 g. of the crude complex were obtained. The filtrate and the collected washings were adjusted to pH 6.2. They were then twice extracted with a volume of petroleum ether and the aqueous phase was extracted with chloroform. The extract was washed with water, dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Addition of petroleum ether to the oil precipitated a residue of g. of a yellow amorphous precipitate containing about of the antibiotic complex. In all 35 g. of crude product, containing 3.5 g. of antibiotic, were obtained.

12 g. of this crude antibiotic (about 10% concentration) were dissolved in 300 cc. of the lower layer of a chloroformzcarbon tetrachloride:methyl alcoholzwater (3:2:4z1) mixture and filtered. The filtrate was adsorbed over a chromatographic column containing 900 g. of silicious acid suspended in chloroform. The column was washed with chloroform and 1 liter fractions were collected. Elution was performed and the samples of the various fractions were analyzed by thin layer chromatography. The fractions 1 to 4 were discarded. Elution was then carried out with chloroform:ethanol mixtures having an increasing percentage of alcohol. The fractions from 5 to were discarded. Eluting with chloroform:ethanol (3:1), eluate fractions 21-26 containined Taimycine C. Eluting with chloroformzethanol (2:1), fractions 27-35 mainly contained the component Taimycine B with lower percentages of Taimycine A and Taimycine C. Eluates with higher percentages of ethanol contained Taimycine The various fractions were collected, concentrated to small volume under reduced pressure, diluted with an equal volume of water, and adjusted to a pH of about 6. By slow evaporation of the residual alcohol, the antibiotics were precipitated as sodium salts from the aqueous concentrate. 130 mg. of Taimycine C were obtained from fractions 21-26. 350 mg. of Taimycine B were obtained from fractions 27-35. 950 mg. of Taimycine A were obtained from fractions 36-50.

The Taimycine C was further purified by crystallization from methanolzwater. 90 mg. of pure product were obtained from the 130 mg. crude. Taimycine B was further purified by distribution in countercurrent in a 40 tube Craig apparatus using phases of cc. each of the chloroform: carbon tetrachloride: methyl alcohol water (2.5 12.5 :4.1) system. At the end, Taimycine B was contained in the tubes 6-17. The mixtures B and A were in the tubes 18-23, Taimycine A was in the tubes 24-3 3.

The three groups of fractions were separately concentrated to small volume, the concentrate was adjusted to pH 6, and then by slowly evaporating off the methyl alcohol, the sodium salts were precipitated. 200 mg. of a mixture containing 60 mg. of Taimycine B and 30 mg. of Tiamycine A were obtained from 340 mg. of crude Taimycine B. The Taimycine A was purified by the same process varying the composition of the mixture used for the countercurrent.distribution (chloroformzoarbon tetrachloridezmethyl alcoholzwater 3:2:4:1), and the number of the transfers which were 100. Starting from 800 mg. of product, there were obtained from the silicious acid column:

150 mg. of Taimycine B from tubes 15-35; 100 mg. of mixture from tubes 36-49; and 420 mg. of Taimycine A from tubes 50-75.

From Taimycine B (sodium salt), the corresponding free acid can be obtained as follows. 5 cc. of N acetic acid were added to a solution containing 300 mg. of Taimycine B in 40 CC. Of QhlQrQfQIm and 10 cc. of etha- 1101. After a few minutes, 45 cc. of water were added. The mixture was stirred and the chloroform layer was then separated. The aqueous layer was extracted with 20 cc. of chloroform. The chloroform extracts were collected together, washed with water and dried over anhydrous sodium sulphate. The chloroform solution was filtered off and concentrated to a volume of 10 cc. After a few hours, 250 mg. of a crystalline precipitate were obtained. Recrystallization from chloroformzethanol gave 200 mg. of Taimycine B melting at 245 C.

In the drawings FIG. 1 shows the ultraviolet absorption spectrum of 'Iaimycine in methanol;

FIG. 2 shows the infrared spectrum of Taimycine A in KBr.

FIG. 3 shows the infrared spectrum of Taimycine B in KBr; and

FIG. 4 shows the infrared spectrum of Taimycine C in KBr.

Also presented are the empirical formulae and elementary analysis of the antibiotics produced in accordance with the present invention.

Empirical formulae and elementary analysis of Taimycine A, B and C Taimycine A (as sodium salt) C H N O Na- /2H O (molecular weight 594.80). Calculated (percent): C, 60.58; H, 7.97; N, 4.71; Na, 3.87. Found (percent): C, 60.30; H, 7.88; N, 4.71; Na, 4.01.

Taimycine B (as free acid) C H N O (molecular weight 526.65). Calculated (percent): C, 68.42; H, 8.04; N, 5.32. Found (percent): C, 68.85; H, 7.81; N, 5.35.

Taimycine C (as sodium salt) C H N O Na (molecular weight 938.12). Calculated (percent): C, 62.70; H, 8.17; N, 4.49; Na, 2.46. Found (percent): 62.50; H, 8.11; N, 4.45; Na, 2.39.

We claim:

1. A microbiological process for the preparation of an antibiotic complex Taimycine and its salts which comprises fermenting the microorganism Streptomyces michiganensis var. amy lolyticus, I.P.V. 1953, in a liquid cultural medium containing an assimilable source of carbon and an assimilable source of nitrogen under aerobic conditions with mineral salts from 24 to 37 C. for from 72 to hours at pH of from 6 to 8.

2. Antibiotic complex Taimycine produced by the process of claim 1.

3. The process of claim 1, wherein the antibiotic complex obtained is isolated from the fermentation broth.

4. The process of claim 3, wherein the antibiotic complex is separated into three components, Taimycine A, Taimycine B and Taimycine C.

5. The process of claim 3, wherein the antibiotic complex produced is converted into free acid form.

6. The antibiotic substance Taimycine A, the sodium salt of which has the formula C H N O Na. /2H O and is characterized by the following properties:

(a) white, amorphous powder (b) elementary analysis: C=60.30%, H=7.88%, N:

(c) melting point: 250 C. (with decomposition) [a] :+7 (c.=0.9* in dioxane); [a] =+24 (c.=0.77 in methanol);

(d) in the UV. spectrum it shows the following absorption maxima:

355 240 and 294 u; Eta-192 and 149 max.

kHClOlENill CHgOH (e) it is soluble in dimethylformamide, dimethylsulphoxide, dioxane, in aqueous alcohols and aqueous acetone; sparingly soluble in lower alcohols, chloroform, acetone, benzene; practiqally insoluble in water and petroleum ether;

13 (f) chromatography on thin layer:

MN Kieselgel G 254, R,=O.60 (butanokacetone:

Water 4:5:1) Kieselgel G, R =0.60 (butanokacetonezwater 4:5:1) Kieselgel HF 254366, R =0.35 (butanol:ace-

tonezwater 4:5:1) MN Kieselgel G 254, R =0.50 (ethyl acetate:

pyridinezisopropyl alcoholzwater 7:2:3z2) Kieselgel G, R =a45 (propanolzethyl acetate:wa-

ter 8:1:1) Alumina, R =Or50 (butanolzpyridinezwater 6:4:3) (g) the IR. spectrum for FIG. 2. 7. The sodium salt of Taimycine A of claim 6. 8. The antibiotic substance Taimycine B and has the formula C H N O in which is characterized by (a) white crystals (b) melting point: 245 C. (c) elementary analysis: C=68.86%; H=7.81%; N:

5.35% (d) UV. spectrum max.

(e) LR. spectrum of FIG. 3 and the sodium salt of which has the following properties:

(a) white amorphous powder (b) elementary analysis: C=67.26%; H=7.97%; N:

(c) melting point: 260 C. (with decomposition) [a] =-|45 (c.=0.9 in dioxane) (d) at the UV. spectrum, it shows the following absorption maxima:

13539 238 and 321 m D:'{;,, =449 and 255 ANaOHMsNiHCHaOH 235 and 320 m E}Z,,,=470 and 250 max.

IHnSQ'QOE N in GHQOH 3225 l 300 14 MN Kieselgel G 254, R =0.5O (ethyl acetate:

pyridine isopropylic alcohol water 7 :2 3 :2) Kieselgel G, R =0.25 (propanokethyl acetatezwater 8:1:1) Alumina, R =0.30 (butano1:pyridine:water 6:4:3) 9. The sodium salt of Taimycine B of claim 8. 10. The antibiotic substance Taimycine C, the sodium salt of which has the formula C H H O Na and is characterized by the following properties:

(a) white amorphous powder (b) elementary analysis: C=62.5O %;H=-8.1l%;N=

(c) melting point 240 C. (with decomposition) [a] =-12 (c.=0.9 in dioxane) (d) at the UV. spectrum it shows the following absorption maxima:

ACHQOH 240 and 290 m E12,, =152 and 157 water 4:5: 1)

Kieselgel G, R;:0.75 (butanol:acetone:water Kieselgel HF 254-366, Rf O-50 (butanolzacetone:

water 4:5 1)

MN Kieselgel G 254, Rf=0-80 (ethyl acetate:

pyridine isopropylic alcohol water 7 2 z 3 2) Kieselgel G, R =0.50 (propanolzethyl acetatezwater 821:1) Alumina, R =0.7O (butanolzpyridinezwater 624:3) the IR. spectrum of Taimycin C is seen in FIG. 4.

11. The sodium salt of Taimycine C of claim 10.

References Cited Derwent Farmdoc #35,097, Abstracting NE68,0751'6, published Dec. 9, 1968.

JEROME D. GOLDBERG, Primary Examiner US. Cl. X.R. 80; 424-122 

